In vivo dynamics of skeletal muscle Dystrophin in zebrafish embryos revealed by improved FRAP analysis

Abstract

Dystrophin forms an essential link between sarcolemma and cytoskeleton, perturbation of which causes muscular dystrophy. We analysed Dystrophin binding dynamics in vivo for the first time. Within maturing fibres of host zebrafish embryos, our analysis reveals a pool of diffusible Dystrophin and complexes bound at the fibre membrane. Combining modelling, an improved FRAP methodology and direct semi-quantitative analysis of bleaching suggests the existence of two membrane-bound Dystrophin populations with widely differing bound lifetimes: a stable, tightly bound pool, and a dynamic bound pool with high turnover rate that exchanges with the cytoplasmic pool. The three populations were found consistently in human and zebrafish Dystrophins overexpressed in wild-type or dmdta222a/ta222a zebrafish embryos, which lack Dystrophin, and in Gt(dmd-Citrine)ct90a that express endogenously-driven tagged zebrafish Dystrophin. These results lead to a new model for Dystrophin membrane association in developing muscle, and highlight our methodology as a valuable strategy for in vivo analysis of complex protein dynamics.DOI: http://dx.doi.org/10.7554/eLife.06541.001

Dystrophin's central domain forms a complex filament that becomes disorganized by in-frame deletions

“…This view is in line with the cooperative behavior of dystrophin repeats observed under unfolding conditions when stretched by optical tweezers (36). This compartmentalization may allow dystrophin to recruit protein partners simultaneously while bound to the plasma membrane (37). In particular, the bulkiest molecules, intermediate filaments and F-actin, are recruited by the large R11-17 domain while microtubules are recruited by R20-23 (38) and due to steric hindrance, these interactions could not occur simultaneously with a straight central domain.…”

Epigenetic Regulators Modulate Muscle Damage in Duchenne Muscular Dystrophy Model

“…Nuclear and dose dependent expression of the transgene was confirmed (Supplementary Figure 1B,C). To test the hypothesis that CBP overexpression improves regeneration, embryos were analysed at 2 dpf, as fibre tips start detaching from somitic borders (Bajanca et al, 2015), and 6 dpf, when regeneration upon damage is underway (Gurevich et al, 2016; Pipalia et al, 2016). While dystrophin -null embryos show characteristic dystrophic muscles from early stages, overexpressing CBP clearly reduced damage not only at later stages but, unexpectedly, also at 2 dpf (Figure 1B).…”

“…Phenolthiourea (0.003%) was added to inhibit pigmentation and facilitate muscle analysis. Dystrophic muscles were characterised by abnormal birefringence, supported by observing disruption of the actin reporter pattern when Tg(actc1b:mCherry) pc4 fish were used, and impaired movement (Bajanca et al, 2015; Berger et al, 2012). The absence of Dystrophin expression in rescued embryos was confirmed by immunohistochemistry.…”

“…CBP expression in zebrafish muscle was reported recently (Batut et al, 2015). We used the zebrafish model, extensively characterised and widely used for studying muscular dystrophy (Bajanca et al, 2015), to explore the effects of overexpressing CBP in a Dystrophin-null background. We have found that overexpressing this single HAT rescues the dystrophic phenotype as efficiently as blocking HDACs.…”

Decision letter: In vivo dynamics of skeletal muscle Dystrophin in zebrafish embryos revealed by improved FRAP analysis

“…DOI: 10.7554/eLife.06541.007 deliberately avoided. The original data and relevant analysis files for each case are available online (Bajanca et al, 2015) to complement representative examples shown in figures and main data summarised in tables. We conclude that, although there is fibre-specific variation of D, there is no evidence that cytoplasmic Dystrophin either binds cytoskeletal elements or is actively transported towards fibre tips.…”